墨兰PI基因过表达载体构建及功能初步分析  

Construction of Overexpression Vector of PI Gene in Cymbidium sinense and Preliminary Analysis of Its Function

作　　者：冯垒 王义琴 韩欣妤 师凯辉 黄鑫 王冬梅 孙博 何玲 朱永平[1] 杨生超 张广辉 和凤美[3] Feng Lei;Wang Yiqin;Han Xinyu;Shi Kaihui;Huang Xin;Wang Dongmei;Sun Bo;He Ling;Zhu Yongping;Yang Shengchao;Zhang Guanghui;He Fengmei(College of Agronomy and Biotechnology,Yunnan Agricultural University,Kunming,650201;Innovation and Utilization of Chinese Medicinal Materials Germplasm in Southwest,Yunnan Agricultural University,Kunming,650201;College of Landscape and Horticulture,Yunnan Agricultural University,Kunming,650201)

机构地区：[1]云南农业大学农学与生物技术学院,昆明650201 [2]云南农业大学西南中药材种质创新与利用国家地方联合工程研究中心,昆明650201 [3]云南农业大学园林园艺学院,昆明650201

出　　处：《分子植物育种》2023年第9期2941-2947,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(32260419);兰科植物保护与利用省创新团队(202105AE160012)共同资助。

摘　　要：MADS-box基因是植物中一类重要的转录调控因子,在生长发育调控和信号传导中发挥着重要作用,并参与花器官的发育和开花时间的调节。兰花中存在大量MADS基因,但这些基因的作用仅有少量研究报道。本研究将墨兰MADS基因家族中PI(PISTILLATA)基因cDNA开放框序列连接到pBWA(V)HU-ccdB-GUS载体上,构建了过表达载体,随后通过农杆菌介导法转化铁皮石斛原球茎,获得转基因株系;采用潮霉素抗性筛选,获得阳性植株,并利用试管开花技术,获得阳性植株开花苗,对开花表型进行观察。结果显示,PI基因在铁皮石斛中过表达能导致铁皮石斛花朵表型发生变化,阳性转化苗花朵在整体花型、中萼瓣和捧瓣的形状、花瓣边缘颜色、合蕊柱内侧的斑点区和紫色条带、舌瓣的颜色和形状等方面都发生了变异,尤其在舌瓣变化最明显,说明PI基因可能与兰花花瓣表型形成有关。本研究结果可为阐明墨兰舌瓣的形成机理及兰花基因工程育种性状改良提供参考依据。The MADS-box gene is an important class of transcriptional regulators in plants.It plays an important role in the regulation of growth and development and signaling,and is involved in the development of floral organs and the regulation of flowering time.There are a large number of MADS genes in orchids,but only a few studies have reported on the role of these genes.In this study,the c DNA open frame sequence of the Cymbidium PI(PISTILLATA)gene was connected On the pBWA(V)HU-ccdB-GUS vector,an overexpression vector was constructed,and then the protocorm of Dendrobium candidum was transformed by Agrobacterium-mediated method to obtain transgenic lines.The hygromycin resistance screen was used to obtain positive plants,and the test tube flowering technique was used to obtain flowering seedlings of positive plants,and the flowering phenotype was observed.The results showed that over-expression of PI gene in De ndrobium candidum can lead to changes in the phenotype of Dendrobium candidum flowers:positive transformed seedling flowers in the overall flower pattern,the shape of the mid-calyx and stem petals,the color of the petal edges,and the spot area inside the pistil variations have occurred in purple bands,the color and shape of tongue flaps,especially in tongue flaps,which shows that the PI gene may be related to the formation of orchid petal phenotype.The results of this study can provide a reference for elucidating the formation mechanism of the Cymbidium tongue flap and improving the genetic engineering breeding traits of orchids.

关 键 词：过表达 MADS-BOX基因 铁皮石斛 遗传转化 试管开花 

分 类 号：S682.31[农业科学—观赏园艺]